Our current corneal research addresses the molecular basis for corneal-specific gene expression, the roles of the abundant, intracellular corneal proteins, the global patterns of corneal gene expression, and stratification of the corneal epithelium. The advances in FY2005 include the following. [unreadable] [unreadable] Mouse aldehyde dehydrogenase 3 (ALDH3A1) comprises half of the water-soluble protein in the corneal epithelial cells. Last year we found that a promoter fragment containing 5? flanking sequences, exon 1, intron 1 and part of exon 2 of the ALDH3a1 gene is activated in co-transfection tests in COS7 cells by Pax6, KLF4, KLF5, Junb and POU2f1/Oct-1. A Pax6 binding site (-110 and -50) and 2 Pou2F1/Oct-1 sites (one in the promoter and one in intron 1) have been found. A position and orientation-dependent negative regulatory element was identified in intron 1 which represses transcription in corneal and other cells. Multiple DNA constructs have been made containing all intronic and 3` sequences of the ALDH3a1 gene to search for regulatory motifs. An explanted rat corneal transfection system is being developed to facilitate study of the regulation of corneal gene promoters in a corneal cell background.[unreadable] [unreadable] In collaboration with Dr. Jerome Kuszak (Rush Presbyterian Hospital, Chicago), we found reductions in microplicae in the corneal surface epithelial cells of ADLH3a1 null mice, indicative of increased cell sloughing. Microarray data indicated that ~50% of the differentially regulated genes in the ALDH3a1 null corneas are those controlled by gamma-interferon. These data suggest that barrier function of the corneal epithelium is compromised causing an immune response. The ALDH3a1 null mice contain Helicobacter (which can lead to ulcers and cancer), while wild type mice do not. Experiments in collaboration with Dr. Vasilis Vasiliou (University of Colorado Health Sciences Center, Denver) using the ALDH3a1 null mice show elevated protein conjugates of 4-hydroxy-nonenal, a substrate of ALDH3A1, consistent with a detoxifying function for this enzyme. One protein, chondromodulin (an anti-angiogenic factor), increases in the cornea of the ALDH3a1 null mice and is located in the cytoplasm and nuclei of the corneal epithelial cells, in contrast to its strictly cytoplasmic presence in wild type mice.[unreadable] [unreadable] We are examining the ocular phenotypes of transgenic mice overexpressing Pax6 and Pax6(5a) transgenes in the cornea. The overexpressing corneas are opaque and enriched with blood vessels. Further studies will involve microarray analysis to identify downstream gene targets of the Pax6 transcription factors being overexpressed. [unreadable] [unreadable] We previously reported that a gelsolin-like protein comprises half of the water-soluble protein of the zebrafish cornea, and that a different gelsolin protein is expressed ubiquitously in the zebrafish at lower levels. In FY2005 in collaboration with Drs. Eugene Koonin (NLM, NIH) and Vasilis Vasiliou (University of Colorado) we established by phylogenetic analysis that the zebrafish corneal-preferred gelsolin-like protein is a scinderin-like protein (also a member of the gelsolin superfamily), while the ubiquitously expressed gelsolin is an authentic gelsolins. Gene duplication has led to 2 scinderin-like genes and 2 gelsolin genes in the zebrafish. The corneal-preferred scinderin-like protein is also expressed in early embryogenesis and contributes to dorsal-ventral signaling. By contrast, the ubiquitous expression of the authentic gelsolin gene is not associated with dorsal-ventral signaling. Thus these closely related genes differ in structure, expression pattern and function. Preliminary results suggest that a 2.5 Kb promoter fragment of the corneal-preferred scinderin-like gene drives the expression of a reporter gene in the embryonic cornea of developing, microinjected zebrafish eggs. Inclusion of exon 1, intron 1 and part of exon 2 in a 4 Kb promoter fragment augments eye expression but does not eliminate it in surrounding tissues. Further constructs are being made including upstream and downstream sequences. [unreadable] [unreadable] Earlier SAGE data on global gene expression in the limbal and central epithelial cells of the adult rat were analyzed in FY2005. The results extend the database of genes expressed in the rodent cornea and suggest an association between several genes (WDNM1-like, mesothelin, and marapsin) that are preferentially expressed in the limbal epithelium with cellular proliferation and migration. [unreadable] [unreadable] Last year, KLF4, the most abundant corneal transcription factor identified by SAGE analysis, was deleted specifically in the cornea of mice using floxed genes (KLF4-loxP mice obtained from Dr. Klaus Kaestner, University of Pennsylvania School of Medicine) and Le-Cre mouse (obtained from Dr. Peter Gruss, Max Planck Institute, Germany). The conditionally deleted KLF4 (Klf4CN) mice had a small eye phenotype with defects in lens, cornea and iris. In FY 2005, we identified and characterized multiple phenotypes in the Klf4CN mice ocular surface and identified KLF4 target genes. Even though the embryonic development of the Klf4CN eyes was normal, the postnatal maturation of the Klf4CN cornea was affected as demonstrated by the presence of only 3-4 epithelial cell layers, swollen, vacuolated basal epithelial and endothelial cells, and edematous stroma. The Klf4CN conjunctiva lacked goblet cells and the anterior cortical lens was vacuolated. Expression of the keratin-12 and aquaporin-5 genes was down regulated, consistent with the Klf4CN corneal epithelial fragility and stromal edema, respectively. We have identified several potential KLF4 target genes in the mouse cornea by microarray analysis. These observations provide new insights into the role of KLF4 in post-natal maturation and maintenance of the ocular surface and suggest that the Klf4CN mouse is a useful model for investigating ocular surface pathologies such as dry eye, Meesmann?s dystrophy and Steven?s-Johnson syndrome.[unreadable] [unreadable] Finally, in FY 2005 we started exploring the role of microRNAs (miRNAs) in mouse corneal development. miRNAs are a class of small endogenous noncoding RNAs that posttranscriptionally down regulate gene expression and play important roles in diverse processes such as embryonic development, differentiation, and cancer. In association with the RNA-induced silencing complex (RISC), miRNAs either cause target mRNA cleavage and degradation or inhibit mRNA translation. We evaluated corneal miRNA expression profiles by microarray analysis in post-natal day 6 (PN6) and 6-week-old (adult) mice. 42 miRNAs demonstrating at least a 2-fold difference in expression between the two developmental stages were identified. Of these differentially expressed miRNAs, miR-184, miR-31, miR-205, and miR-204 were corneal-enriched and their expression increased in adult corneas compared to PN6 corneas. miR-184 is the most abundant miRNA in adult mouse cornea. The gene for miR-184 is located in an intergenic region on mouse chromosome 9. Preliminary data from 2-dimensional gel electrophoresis corneal proteins indicate differences in protein levels between PN9 and adult corneas. These data will be used in conjunction with SAGE and microarray data to identify miRNA target genes.